Directed differentiation and maturation of pluripotent cells into hepatocyte like cells by modulation of Wnt-signalling pathway

ABSTRACT

Provided are improved methods using Glycogen synthase kinase 3 (GSK3) inhibitors by which endodermal cells, notably endodermal cells derived from human pluripotent stem cells (hPS), such as but not limited to hiPS-cells and hES-cells may be differentiated into hepatocyte like cells. The specific modulation of wingless integration gene (WNT)-signalling pathway and use of GSK3 inhibitors achieve direct differentiation and maturation of hepatocytes derived from human pluripotent stem (hPS) cells. GSK-3 inhibitors, when added to the growth medium at certain developmental stages, leads to more mature and functional features for the hepatocyte like cells as well as more pure and homogenous populations of hepatocyte like cells. Provided are also hepatocyte like cells obtained by these methods as well as compositions comprising them.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/636,543, filed on Jan. 2, 2013, now U.S. Pat. No. 9,394,522, issuedon Jul. 19, 2016, which is a U.S. National Stage patent applicationpursuant to 35 U.S.C. § 371 of International Patent ApplicationPCT/EP2001/001411, filed on Mar. 22, 2011, and published as WO2011/116930 on Sep. 29, 2011, which claims priority to U.S. ProvisionalPatent Application Ser. No. 61/316,021, filed on Mar. 22, 2010, andDenmark Patent Application PA 2010 00234, filed on Mar. 22, 2010, all ofwhich are incorporated herein by reference in their entireties for allpurposes.

TECHNICAL FIELD

The present invention relates to modulation of the wingless integrationgene (Wnt)-signalling pathway to achieve directed differentiation andmaturation of hepatocytes derived from human pluripotent stem (hPS)cells. Furthermore, the invention refers to the use of a glycogensynthase kinase 3 (GSK3) inhibitor for activation of the Wnt pathwaywhen the cells are at certain developmental stages during theorganogenesis. The inventors have, as disclosed herein, found that GSK-3inhibitors, when added to the growth medium at certain developmentalstages leads to more mature and functional features for the hepatocytelike cells as well as more pure and homogenous populations of hepatocytelike cells, compared to currently available state of the art methods.

BACKGROUND OF THE INVENTION

Human pluripotent stem cells (hPS) are expected to revolutionize theaccessibility to a variety of human cell types. The possibility topropagate pluripotent human embryonic-derived stem (hES) cells and humaninduced pluripotent stem (hiPS) cells and subsequently differentiatethem into the desired target cell types will provide a stable andvirtually unlimited supply of cells for a range of applications in vivoand in vitro.

Liver failure and end-stage liver diseases are responsible for a hugeamount of deaths around the world and is a major burden on the healthcare system. Liver transplantation remains the most successfultreatment. However, the efficacy of this procedure is limited andconnected to many complications such as infection or rejection. Livertransplantation also suffers from shortage of available donor organs andthe treated patients will very often be referred to lifelongimmunosuppression therapy. By reducing the need for organs, cell-basedtreatment will be of great importance to both society and to theindividuals suffering from these severe diseases.

Furthermore, the liver is the centre of metabolism and detoxification inthe human body, and therefore huge efforts have been undertaken in orderto identify a reliable source of functional cell types for in vitrotesting. Unfortunately, the complexity and function of the liver is notmirrored by any cell type available today. The availability of primaryhuman liver cells is very limited and the cells are also known torapidly loose their normal phenotype and functional properties when usedfor in vitro applications. One often used alternative to primary cellsare hepatic cell lines which in turn contain very low levels of (ortotally lack) metabolising enzymes and have distributions of otherimportant proteins substantially different from the native hepatocyte invivo. Thus, many tests are still performed using animal material, eventhough liver metabolism is known to be species specific and therebygenerating difficulties in predicting liver metabolism and toxicity inother species than the one tested.

In pharmaceutical development, adverse liver reactions remain the mostprominent side effect. Therefore early prediction of human livertoxicity liabilities is of paramount importance when selecting compoundsto enter clinical trials. Efforts to improve capabilities in this areamust address both the availability question and development of models,which provide greater coverage for the complex biological processeswhich coincide to induce adverse liver injury in humans.

Accordingly there is an urgent need for a model system that mimics humanliver cells and that is able to predict effects of candidate moleculesin the development of new drugs or chemicals. Regarding bothavailability and physiological relevance, hPS cells may serve as anideal renewable source of functional human hepatocytes.

During the embryogenesis and the formation of the yolk sac and theplacenta, two types of endoderm cells form: the extraembryonic endodermcells and the definitive endoderm (DE) cells. Extraembryonic endodermarises at the blastocyst stage and eventually forms two subpopulations:visceral endoderm and parietal endoderm. Extraembryonic endoderm cellsshare the expression of many genes with definitive endoderm (DE) cells(cells that give rise to the endodermal organs), including the oftenanalyzed transcription factors Sox17 (Kanai-Azuma et al., 2002), FoxA1and HNF3b/FoxA2 (Belo et al., 1997; Sasaki and Hogan, 1993). However,some markers are expressed in both mesoderm and definitive endoderm,such as CXCR4. Those commonly expressed markers can be used incombination with Sox17 and FoxA2 to type DE cells. Sox7 is a marker onlyexpressed in extraembryonic endoderm.

The definitive endoderm cells give rise to endodermal organs and thushepatic cell types. However, early endoderm development is not wellunderstood. Directed studies of cultured mouse embryos (Lawson et al.,1986, 1991; Lawson and Pedersen, 1987) have revealed that DE begins toform at the embryonic days 6-6.5 (E6-6.5) and that by the end ofgastrulation (E7.5), some cells only give rise to endodermalderivatives. It is not known whether the initial DE cells aremultipotent. Fate mapping studies (Lawson et al., 1991; Tremblay andZaret, 2005) suggest that the first endoderm cells that migrate throughthe primitive streak (PS) at E6.5 are fated to become liver, ventralpancreas, lungs and stomach. Co-culture experiments show that theendoderm at this state is not fully committed at the early state ofdevelopment (Wells and Melton, 2000).

For in vitro purposes, for example D'Amour et al. and Hay et al. havedeveloped protocols for deriving definitive endoderm from hES cells(D'Amour et al., 2005; D'Amour et al., 2006, Hay et al., 2007; Hay etal., 2008) as well as protocols for derivation of hepatic endoderm fromhiPS cells (Hay et al. 2010).

The Wnt pathway describes a series of events that occur when Wntproteins bind to cell-surface receptors of the Frizzled family, causingthe receptors to activate other proteins and ultimately resulting in achange in the amount of β-catenin that reaches the nucleus. Amembrane-associated Wnt receptor complex will, when activated by Wntbinding, inhibit a second complex of proteins that includes e.g. theproteins GSK3 and axin. This complex normally promotes the proteolyticdegradation of the β-catenin intracellular signalling molecule. Afterthis inhibition, a pool of cytoplasmic β-catenin is presentintracellularly, and some β-catenin is able to enter the nucleus andinteract with transcription factors to promote specific gene expression.The Wnt/β-catenin signalling regulates key physiological events inherentto the liver including development, regeneration and development ofcancer, by dictating several biological processes such as proliferation,apoptosis, differentiation, adhesion, zonation and metabolism in variouscells of the liver (Nejak-Bowen et al., 2008).

Crosstalk between mesoderm and endoderm is required for liverdifferentiation. The wnt signalling molecule, Promethues/Wnt2b has beenshown to be expressed in the discrete lateral plate mesoderm adjacent tothe endoderm that will become hepatic endoderm in the zebra fish.Morpholino antisence knock-down of the wnt-gene will obliterate orreduce the early hepatic differentiation markers of the hepaticendoderm, hHEX and Prox1, indicating a role for wnt signalling inhepatic cell fate specification. In addition inhibition of beta-cateninsignalling in the zebrafish embryo strongly reduced the development ofhepatic tissue, suggesting a role for the canonocal/beta-catening Wntsignalling in liver cell fate specification. (Ref. Elke Ober et al.Nature 442, 688-691 (10 Aug. 2006) However, observations in xenopussuggests that wnt signalling is crucial for patterning of the definitiveendoderm into the anterior and posterior endoderm, where inhibition ofwnt signalling leads to the anterior endoderm and hepatic inductionwhile wnt signalling leads to posterior endoderm and intestinalinduction. However, just after anterior patterning wnt signalling isimportant for hepatic induction and delamination of hepatoblasts fromthe hepatic endoderm. The data from the zebra fish and xenopus aresomewhat contradictory and represent the complexity of Wnt-signalling inearly hepatic differentiation. Fine tuning and timing of non active andactive Wnt signalling seem to be important for early hepaticdifferentiation.

The use of GSK inhibitors have previously been described for earlydifferentiation towards endoderm. WO08094597 (Dalton) describes a methodof producing mesendodermal from primate pluripotent stem cells (pPSC) bycontacting the pPSC with an effective amount of GSK inhibitor in adifferentiation media.

WO2007050043 (Stanton) describes a method for producing a mesodermal oran endodermal cell from a pluripotent stem cell, comprising aWnt-signalling pathway in the pluripotent stem cell.

US2006003446 (Keller) describes a way of making a cell populationenriched for endoderm cells culturing embryonic stem cells in theabsence of serum and in the presence of activin and an inhibitor ofWnt-signalling.

US 20100062527 (Pera et al.) describes in Example 3 culturing of HES2 or3 cells “for 5 days on MEF feeders with 20% FCS hES medium in organculture dishes. 20% FCS hES medium was replaced with 3i medium and cellswere keeping in culture in this medium for 3 days. Cells were detachedas clumps by collagenase and feeder cells were removed by sedimentationin DMEM/F-12 medium. Cells were seeded on Matrigel-coated organ culturedish as 1 to 1 split and culture with 3i medium. After 3 to 5 days inculture hepatoblast-like cells appeared. They were subsequentlypropagated using 3i of Kubota's medium following enzymatic dissection.”The 3i medium contains: Neural basal medium 50%, DMEM/F-12 50%, N2supplement 1/200 v/v, B27 supplement 1/100 v/v, 100 mM L-glutamine 1/100v/v, 0.1 M beta-ME 1/1000 v/v, SU5402 (FGFR inhibitor) 2 μM, PD184352(ERK cascade inhibitor) 0.8 μM, and CHIR99021 (GSK3 inhibitor) 3 μM.

SUMMARY OF THE INVENTION

Present invention describes improved methods by which endodermal cells,notably endodermal cells derived from human pluripotent stem cells(hPS), such as but not limited to hiPS-cells and hES-cells may bedifferentiated into hepatocyte like cells. Further, the inventionrelates to new ways of in vitro stimulation of the organogenesis of theliver which is crucial for development of a mature and functionalproduct; the hepatocyte-like cells derived from human pluripotent stemcells.

As disclosed herein, the potential roles of Wnt/β-catenin signallingduring the phases of liver development, including competence, hepaticinduction, expansion and morphogenesis have been examined and exploitedin order to obtain improved hepatocyte like cells from human pluripotentcell types.

The present invention is the first to describe and exploit the specificmodulation of Wnt-signalling and the use of a GSK inhibitor for hepaticdifferentiation using endoderm cells as starting material, i.e. focusingon the direction and modulation of a later stage of the organogenesiscompared to previous publications, which are focusing on the use of aninhibitor during differentiation up to the definitive endoderm (DE)stage.

In the present invention an hPS cell derived hepatocyte-like cellpopulation is generated using novel strategies by affecting the Wntsignalling. The cells obtained by the method as described herein byusing a GSK3 inhibitor show improved morphology, higher purity andenzymatic activity compared to cells obtained without the use of a GSK-3inhibitor, and therefore offers improved applicability for drugdiscovery purposes and regenerative medicine. Furthermore the cells areshowing a stable expression of important liver-expressed marker genessuch as Albumin, CYP1A2, CYP3A4, UGT2B7, GSTA1 as well as drugtransporters for an extended period of time compared to cells obtainedwithout the use of GSK3 inhibitors and are thus highly suitable for drugdiscovery purposes and regenerative medicine.

We have found that addition of a GSK3 inhibitor helps to generate apure, synchronized, and metabolically induced culture of hPS derivedhepatic like cells both on feeder and in feeder free conditions. In theexamples herein feeder free conditions have been demonstrated.

A particular finding is that addition of a GSK inhibitor after initialdifferentiation of the hPS-cells into mesendodermal progenitors orbeyond improves to the further differentiation and quality of thehepatocyte like cells by stimulating an increased cell type homogeneity,catalytic activity and increased expression of hepatic markers.

The present invention disclose how the use of GSK inhibitors and/ormodulation of the Wnt signalling pathway at certain time points duringthe differentiation period help to ensure improved directeddifferentiation revealed by increased enzyme activity, pronounced geneexpression and increased homogeneity of the obtained hepatocyte likecells.

As illustrated herein, a number of different protocols, includingdifferent growth media and incubation times are tested and assessed inrelation to their applicability to stimulate directed differentiation ofundifferentiated pluripotent cells into hepatocyte like cells.

DETAILED DESCRIPTION OF THE INVENTION

Present invention relates to the use of a GSK-3 inhibitor for directeddifferentiation of endodermal cells into hepatocyte like cells. Thestarting material in present invention relates to any pluri- ormultipotent human cell, developed to any stage at or beyond theendodermal stage, such as hepatic endoderm, including extraembryonic anddefinitive endoderm cells or hepatic progenitors. Thus, the endodermalcells may comprise definitive endoderm, extraembryonic endoderm orhepatic progenitors.

Further, the GSK-3 inhibitor may be used together with a histonedeacetylase (HDAC) inhibitor such as e.g. sodium butyrate (NaB).

The present invention relates to improved differentiation of pluripotentcells (e.g., human induced pluripotent stem cells (hiPS) or humanembryonic stem cells (hESC) cultured under both feeder free conditionsand in the presence of feeders, in which the Wnt signalling pathway isinfluenced by the addition of bioactive compounds to the growth mediumafter initial differentiation. Initial differentiation may in thiscontext be considered as differentiation from pluripotent stem cellsinto cells resembling the definitive endoderm, the mesendoderm and/orcells resembling hepatic progenitors.

As exemplified below, the GSK3 inhibitor may be added at predeterminedtime-points during a differentiation protocol, but more importantly thetiming of GSK-3 inhibitor addition is to be determined by thedevelopmental stage of the growing cells, such as after differentiationof the endoderm cells into hepatic progenitors.

Thus, the GSK-3 inhibitor is present after initial differentiation intoendodermal cells as extraembryonic or definitive endodermal cells. GSK-3inhibitors are useful in all aspects of the invention which relate tothe differentiation and maturation of hepatocytes from hPS cells andpurification in hepatic endoderm differentiation. They are used inconcentrations of about 0.001 to about 100 μM or more, about 0.05 toabout 75 μM, about 0.1 to about 50 μM, about 0.25 to about 35 μM, about0.5 to about 25 μM. In the case of the use of BIO, this GSK inhibitor isused in the differentiation medium in an amount ranging from about 0.05to about 50 μM, about 0.1 to about 10 μM, about 0.5 to about 5 μM, about1-3 μM. In the case of SB216763 the GSK inhibitor is used in thedifferentiation medium in an amount ranging from about 30 nM to about 15μM, about 30 nM to about 1 μM, about 1 μM to about 5 μM, about 5 μM toabout 15 μM. In the case of Kenpaullone the GSK inhibitor is used in thedifferentiation medium in an amount ranging from about 30 nM to about 20μM, about 30 nM to about 1 μM, about 1 μM to about 5 μM, about 5 μM toabout 15 μM. In the case of Indirubin-3′-oxime the GSK inhibitor is usedin the differentiation medium in an amount ranging from about 30 nM toabout 15 μM, about 30 nM to about 1 μM, about 1 μM to about 4 μM, about5 μM to about 10 μM.

Thus, in one embodiment, the present invention relates to a method fordifferentiating definitive endodermal cells to hepatocyte like cells byadding a GSK-3 inhibitor to the growth medium. Depending on the growthmedia composition and developmental stage of the cells, the growth mediamay be changed or components added in order to direct differentiationtowards a hepatocyte like cell type. Because the developmental stage ofthe cells is crucial for the timing of GSK-3 inhibitor addition, thechronology in the exemplary protocols may differ. In the tables beloware listed examples of protocols in which growth factors and GSK-3inhibitors are added.

As shown in the Examples herein, it seems advantageous to add a GSK-3inhibitor at a later stage of differentiation to avoid massive celldeath. Moreover the resulting purity of the cell populations (FIGS.8A-E, purity table) also suggest that, at least for BIO, addition at alater stage (day 10+) gives greater final purity.

Using endoderm as starting material, the GSK-3 inhibitor may be addedimmediately at the start of culturing and throughout the duration of theprotocol as illustrated in Scheme A-G. Hence a method for thepreparation of hepatocyte like stem cells according to present inventionmay resemble any of the schemes as illustrated in scheme A-G below.

Scheme A Starting material Time, day Liquid medium comprising: DE-cells0 to 2-8 Medium A1 + GSK-3 inhibitor, e.g. BIO 2-8 to 10-20 Medium A2 +GSK-3 inhibitor, e.g. BIO 10-20 to 45 Medium A3 + GSK-3 inhibitor, e.g.BIO

Scheme B Starting material Time, day Liquid medium comprising: DE-cells0 to 2-8 Medium B1 + GSK-3 inhibitor, e.g. BIO 2-8 to 10-20 Medium B2 +GSK-3 inhibitor, e.g. BIO 10-20 to 45 Medium B3

Scheme C Starting material Time, day Liquid medium comprising: DE-cells0 to 2-8 Medium C1 2-8 to 10-20 Medium C2 + GSK-3 inhibitor, e.g. BIO10-20 to 45 Medium C3 + GSK-3 inhibitor, e.g. BIO

Scheme D Starting material Time, day Liquid medium comprising: DE-cells0 to 2-8 Medium D1 + GSK-3 inhibitor, e.g. BIO 2-8 to 10-20 Medium D210-20 to 45 Medium D3 + GSK-3 inhibitor, e.g. BIO

The growth media as listed in scheme A-D may further comprise thefollowing components:

Examples of Growth media Comprise further components that may be addedA1, B1, C1, Activin A RPMI1640 + PEST + glutamax D1 B27 Activin NaB A2,B2, C2, Vitrohes D2 1% DMSO A3, B3, C3, HGF WME + SQ (−GA1000) +glutamax + D3 dexamethasone PEST OsM Dexamethasone bFGF HGF DMSONicotinamide ITS Glucagon

Additionally as specified in scheme E-G, when using DE-cells as startingmaterial, other media and culturing intervals may be used to facilitatedirected differentiation to hepatocyte like cells.

Scheme E Starting material Time, day Liquid medium comprising: DE-cells0 to 3-14 Medium E1 3-14 to 45 Medium E2 + GSK-3 inhibitor, e.g. BIO

Scheme F Starting material Time, day Liquid medium comprising: DE-cells0 to 1-5 Medium F1 1-5 to 6-14 Medium F2 6-14 to 45 Medium F3 + GSK-3inhibitor, e.g. BIO

Scheme G Starting material Time, day Liquid medium comprising: DE-cells0 to 1-5 Medium G1 1-5 to 6-14 Medium G2 + GSK-3 inhibitor, e.g. BIO6-14 to 45 Medium G3 + GSK-3 inhibitor, e.g. BIO

The growth media as listed in scheme E-G may further comprise thefollowing components

Growth Examples of media Comprise further components that may be addedE1, F1, aFGF WME + SQ (−GA1000) + glutamax + PEST G1 bFGF OsM BMP2 DMSOBMP4 Nicotinamide Dexamethasone ITS HGF Glucagon FBS F2, G2 DMSOVitrohes E2, F3, HGF WME + SQ (−GA1000) + glutamax + PEST G3Dexamethasone OsM HGF DMSO Glucagon

When using hepatic progenitors as starting material, schemes H-J asspecified below, may be used to direct differentiation into hepatocytelike cells. As shown in table H-J, a method for the preparation ofhepatocyte like stem cells according to present invention may resembleany of the schemes as illustrated in scheme H-J below

Scheme H Starting material Time, day Liquid medium comprising: Hepatic 0to 10-20 Medium H1 + GSK-3 inhibitor, e.g. BIO progenitors 10-20 to 45Medium H2 + GSK-3 inhibitor, e.g. BIO

Scheme I Starting material Time, day Liquid medium comprising: Hepatic 0to 10-20 Medium I1 + GSK-3 inhibitor, e.g. BIO progenitors 10-20 to 45Medium I2

Scheme J Starting material Time, day Liquid medium comprising: Hepatic 0to 10-20 Medium J1 progenitors 10-20 to 45 Medium J2 + GSK-3 inhibitor,e.g. BIO

The growth media as listed in scheme H-J may further comprise thefollowing components

Growth Examples of further components that may be media comprise addedH1, I1, J1 DMSO Vitrohes H2, I2, J2 HGF WME + SQ (−GA1000) + glutamax +PEST Dexamethasone OsM HGF DMSO Glucagon

As used in schemes A-J and corresponding growth media, the componentsmay be added in the following concentrations:

Activin A: 5-250 ng/ml, such as e.g. 50-200 ng/ml such as 75-150 ng/mlpreferably 100 ng/ml

GSK-3 inhibitor: 0.1-10 μM such as e.g., 1-5 μM preferably 1.4 μM and3.5 μM for late stage media (A3, B3, C3, D3 etc) and early stage media(A1, A2, B1, B2, C1, C2, D1, D2 etc) respectively. Meaning a 2-3 timeshigher concentration added for the use in the early stage media, than inlate stage media.

Dexamethasone: 0.01-5 μM such as eg. 0.05-2 μM preferably 0.1 μM

HGF (human growth factor): 1-50 ng/ml such as 5-30 ng/ml preferably 20ng/ml

aFGF (acidic fibroblast growth factor): 10-250 ng/ml such as eg. 50-200ng/ml preferably 100 ng/ml

bFGF. (basic fibroblast growth factor): 1-25 ng/ml such as eg. 5-10ng/ml, preferably 5 ng/ml

BMP2 (bone morphogenic protein 2) 10-250 ng/ml, such as eg 25-100 ng/mlpreferably 50 ng/ml

BMP4 (bone morphogenic protein 4): 25-500 ng/ml such as eg. 50-250 ng/mlpreferably 200 ng/ml

Glucagon; 0.3-20 ng/ml, such as eg. 1-10 ng/ml, such as eg. 2-5 ng/mlpreferably 3 ng/ml

DMSO (dimethyl sulfoxide): 0.05-5% such as eg. 0.1-2% preferably 0.5%

OsM (Ocostatin M): 1-25 ng/ml, such as eg. 5-15 ng/ml preferably 10ng/ml

PEST: 0.01-5%, preferably 0.1%

Nicotinamide: 1-25 mM, such as eg. 5-15 mM, preferably 10 mM

ITS (Insulin-Transferrin-Selenium-G Supplement (100×)): 1-25 μl/ml, suchas eg. 5-15 μl/ml preferably 10 μl/ml

FBS (fetal bovine serum): 0.1-10%, such as 0.5-5%, such as eg. 1-4,preferably 2%

B27 (B-27 Serum-Free Supplement (50×), liquid (Invitrogen)): preferablya dilution to 1×

NaB (Sodium Butyrate): 0.1-10 mM, such as 0.5-5 mM, preferably 1 mM

RPMI1640 ([Sigma-Aldrich] bicarbonate-buffered, defined cell culturemedium, must be supplemented with glutamine 0.3 g/L, L-L-Glutamine(glutamax)

VitroHES ([Vitrolife]) defined, balanced cell culture medium for thesupport of human embryonic stem cell culture. Must be supplemented withbFGF at preferably at a concentration of between 1 ng/ml-100 ng/ml

WME+SQ medium-Williams Medium E supplemented with SQ medium kit (OsM,Insulin, HGF, EGF)

Specific examples are also given in the Examples herein.

Improved differentiation in present invention is intended to includebetter maturation, improved cell type homogeneity, increased enzymaticactivity or substrate conversion, assessed by comparing the cellsobtained by a method using a GSK-3 inhibitor as disclosed herein, tocells obtained without the use of a GSK-3 inhibitor. Further, improveddifferentiation may be improved expression of genes associated with ahepatic cell fate assessed by comparing the cells obtained by a methodusing a GSK-3 inhibitor as disclosed herein, to cells obtained withoutthe use of a GSK-3 inhibitor. However, improved differentiation asdisclosed herein also intended to include improved viability, ability toengraft and better applicability for example drug screening and drugdevelopment purposes.

Manipulation of the Wnt-signalling pathway by the use of a GSK-3inhibitor has shown to improve the gene-expression profiles ofhepatocyte like cells.

hESC-HEPs may be derived from a xeno-free hPS cell line which wasestablished under animal-free conditions. Moreover, hESC-HEPs may bederived from such an hPS cell line under xeno-free (animal free)conditions, giving rise to a truly xeno-free cell composition. Such acell line would be better suited to therapeutic or regenerative medicineapplications and could be distinguished from a non-xeno-free hESC-HEPline by the presence in non-xeno-free lines of the non-human sialic acidNeu5Gc or other non-human markers (Martin M J et al 2005).

The products obtained according to present invention comprise cellsobtained by the method or schemes as disclosed herein, in vitro derivedhepatocyte-like cells growing in the presence of a GSK-3 inhibitor (e.g.BIO) and/or a HDAC inhibitor (e.g. NaB) or compositions comprising invitro derived hepatocyte-like cells and a GSK-3 inhibitor (e.g. BIO)and/or a HDAC inhibitor (e.g. NaB).

Further the compositions as claimed in present invention relates tocompositions of in vitro derived human cells comprising hepatocyte likecells wherein at least 70% such as e.g. 75%, 80%, 90% or 95% of thecells are hepatocyte like cells

The compositions according to present invention also relates tocompositions wherein the hepatocyte like cells show cytochrome P450activities exceeding a fold change of at least 10, such as e.g. 13, suchas e.g. 18 in cytochrome P450 activity, when compared to cultures wherea GSK inhibitor is not used. The cytochrome P450 activity may bemeasured by cytochrome P450 1A.

Compositions according to present invention may further show elevatedexpression of hepatocyte-associated genes such as e.g. CYP1A1, CYP1A2,CYP3A4, CYP2C9, CYP7A1, MRP2. They may also show increased metabolicactivity, as evidenced by increased activity of UGT enzymes(UDP-glucuronyltransferases) such as UGT1A1, UGT1A6, UGT1A9, UGT2B7).Improved metabolic activity may also be shown by ability ofhepatocyte-like cell compositions to metabolise drugs such asparacetamol and Diclofenac.

The cells obtained according to the methods and principles as laid outin present invention may be used to a multitude of purposes comprisingdrug discovery processes, toxicity test, for studying drug transporters,drug metabolizing enzyme, as in vitro models for studying hepatogenesis,such as, e.g., early hepatogenesis. for studying humanhepatoregenerative disorders, for in vitro hepatotoxicity testing.Further the hepatocyte-like cells obtained according to the directionsgiven in present invention may be used for therapeutic purposescomprising: in a medicament, for the manufacture of a medicinal productfor the prevention and/or treatment of pathologies and/or diseasescaused by tissue degeneration, such as, e.g., the degeneration of livertissue, for the manufacture of a medicinal product for the treatment ofliver disorders or for the manufacture of a medicinal product for theprevention and/or treatment of liver disorders selected from the groupconsisting of auto immune disorders including primary biliary cirrhosis;metabolic disorders including dyslipidemia; liver disorders caused bye.g. alcohol abuse; diseases caused by viruses such as, e.g., hepatitisB, hepatitis C, and hepatitis A; liver necrosis caused by acute toxicreactions to e.g. pharmaceutical drugs; and tumour removal in patientssuffering from e.g. hepatocellular carcinoma. Alternatively, the cellsobtained according to the directions provided in present invention maybe used for the manufacture of a medicinal product for the treatmentand/or prevention of metabolic pathologies and/or diseases, forobtaining metabolically improved hepatocyte-like cells, for studyingmaturation towards hepatocyte-like cells or for screening a compound forits ability to modulate hepatocellular function, comprising exposing invitro derived hepatocyte-like cells obtained according to the directionsprovided herein to the compound, determining any phenotypic or metabolicchanges in the cells that result from contact with the compound, andcorrelating the change with an ability to modulate hepatocellularfunction.

The present invention also relates to a method for the preparation ofhepatocyte-like cells and to compositions containing such cells. Detailsappear from the appended claims. The particulars and details describedabove apply mutatis mutandis to all aspects of the invention.

DEFINITIONS

As used herein, “human pluripotent stem cells” (hPS) refers to cellsthat may be derived from any source and that are capable, underappropriate conditions, of producing human progeny of different celltypes that are derivatives of all of the 3 germinal layers (endoderm,mesoderm, and ectoderm). hPS cells may have the ability to form ateratoma in 8-12 week old SCID mice and/or the ability to formidentifiable cells of all three germ layers in tissue culture. Includedin the definition of human pluripotent stem cells are embryonic cells ofvarious types including human embryonic stem (hES) cells, (see, e.g.,Thomson et al. (1998), Heins et. al. (2004), as well as inducedpluripotent stem cells (see, e.g. Yu et al., (2007) Science 318:5858);Takahashi et al., (2007) Cell 131(5):861). The various methods and otherembodiments described herein may require or utilise hPS cells from avariety of sources. For example, hPS cells suitable for use may beobtained from developing embryos. Additionally or alternatively,suitable hPS cells may be obtained from established cell lines and/orhuman induced pluripotent stem (hiPS) cells.

As used herein “hiPS cells” refers to human induced pluripotent stemcells.

As used herein “definitive endoderm (DE)” and definitive endoderm cells(DE-cells) refers to cells exhibiting such as but not limited to proteinor gene expression and or/or morphology typical to cells of thedefinitive endoderm or a composition comprising a significant number ofcells resembling the cells of the definitive endoderm.

As used herein, “hepatic progenitors” or “hepatic progenitor cells”refers to refers to cells exhibiting markers such as but not limited toprotein or gene expression and/or morphology typical to cells of thedefinitive endoderm or a composition comprising a significant number ofcells resembling the cells of the hepatic progenitors.

As used herein, “hepatocyte-like cells (HCLC)” is intended to mean acell type which is expressing mature hepatic markers such as Albumin,CYP3A4, UGT2B7, OATP-2.

As used herein, “hESC-HEP” is intended to mean a cell type derived fromhuman embryonic stem cells which is expressing mature hepatic markerssuch as Albumin, CYP3A4, UGT2B7, OATP-2, ADH1A, UGT1A6, CYP2C9, CYP2C19and CYP2D6.

As used herein, “Wnt-signalling” refers to the pathways included in theWnt signalling as reviewed in, but not limited to, Lade, A G, and Monga,S P, Dev. Dyn. 240:486-500 (2011).

As used herein HDAC inhibitors refers to Histone deacetylase inhibitors.

As used herein, “GSK inhibitor” refers to a compound which inhibits GSK(especially GSK3, including GSK3alpha or GSK3beta). Examples ofpreferred GSK inhibitors for use in the present invention include one ormore of the following:

BIO (2′Z,3′E)-6-Bromoindirubin-3′-oxime (GSK3 Inhibitor IX);

BIO-Acetoxime (2′Z,3′E)-6-Bromoindirubin-3′-acetoxime (GSK3 InhibitorX);

(5-Methyl-IH-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine(GSK3-Inhibitor XIII);

Pyridocarbazole-cyclopenadienylruthenium complex (GSK3 Inhibitor XV);

TDZD-8 4-Benzyl-2-methyl-I,2,4-thiadiazolidine-3,5-dione (GSK3betaInhibitor I);

2-Thio(3-iodobenzyl)-5-(I-pyridyl)[I,3,4]-oxadiazole (GSK3beta InhibitorII);

OTDZT 2,4-Dibenzyl-5-oxothiadiazolidine-3-thione (GSK3beta InhibitorIII);

alpha-4-Dibromoacetophenone (GSK3beta Inhibitor VII);

AR-AO 14418 N-(4-Methoxybenzyl)-N′-(5-nitro-I,3-thiazol-2-yl)urea(GSK-3beta Inhibitor VIII);

3-(I-(3-Hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione(GSK-3beta Inhibitor XI);

TWSI 19 pyrrolopyrimidine compound (GSK3beta Inhibitor XII);

L803 H-KEAPPAPPQSpP-NH2 or its Myristoylated form (GSK3beta InhibitorXIII); and

2-Chloro-I-(4,5-dibromo-thiophen-2-yl)-ethanone (GSK3beta Inhibitor VI);

Aminopyrimidine CHIR99021.

Kenpaullone (9-Bromo-7,12-dihydro-indolo[3,2-d][1]benzazepin-6(5H)-one,

SB2167633-(2,4-Dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dioneand Indirubin-3′-monoxime

Furthermore small molecules can be used to direct Wnt-signalling. Aswell GSK3β blockers for Wnt-signalling induction can be used formodulation of Wnt-signalling to achieve directed differentiation andmaturation. The Wnt-signalling pathway can be induced at a later stageafter initiation or before induction occurs.

As used herein “CYP” is intended to mean Cytochrome P, and morespecifically Cytochrome P 450, the major phase I metabolizing enzyme ofthe liver constituting of many different isoenzymes, such as CYP1A1,CYP1A2, CYP1B1, CYP2A6/2A7/2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C19,CYP2D6, CYP2E1, CYP3A4, CYP3A5, CYP3A7 and CYP7A1.

As used herein, the term “GST” is intended to mean glutathionetransferase, and examples of subtypes thereof are GST A1-1, GST M1-1,and GST P1-1.

As used herein the term “UGT” is intended to mean uridinediphosphoglucuronosyltransferase, which is a group of liver enzymescatalyzing glucuronidation activities.

By the term “functional drug metabolising enzymes” is intended to meanfunctional enzymes belonging to the phase I and phase II enzymes thatperform chemical modifications of xenobiotics and drugs, so called drugor xenobiotic metabolism.

As used herein, the term “functional activity” means effectivemeasurable hepatic cell function, such as a measurable transportation ofdrugs for drug transporters and a measurable metabolism of enzymes forthe Cytochrome P450s (CYPs), commonly detected in primary humanhepatocytes.

As used herein, the term “extraembryonic endoderm (ExE)” is intended tomean the differentiated endodermal cells that, as to the opposite of thedefinitive endoderm, will constitute the compartments outside the embryoin the human development, such as the yolk sac.

As used herein, the term “AAT” is intended to mean the liver markeralpha-anti-trypsin.

As used herein, the term “AFP” is intended to mean the liver markeralpha-fetoprotein. As used herein, the term “BSEP” is intended to meanthe bile transporter bile salt export pump.

As used herein, the term “CK” is intended to mean the liver markercytokeratin (used interchangeably) with different subtypes such asCytokeratin 18 (CK18/KRT18), Cytokeratin 19 (CK19/KRT19), Cytokeratin 8(CK8) and Cytokeratin 7 (CK7).

As used herein, the term “FGF” means fibroblast growth factor,preferably of human and/or recombinant origin, and subtypes belongingthereto are e.g. “bFGF” (means basic fibroblast growth factor, sometimesalso referred to as FGF2) and FGF4. “aFGF” means acidic fibroblastgrowth factor (sometimes also referred to as FGF1).

As used herein, the term “BMP” means Bone Morphogenic Protein,preferably of human and/or recombinant origin, and subtypes belongingthereto are e.g. BMP4 and BMP2.

As used herein, the term “HGF” means Hepatocyte Growth Factor,preferably of human and/or recombinant origin.

As used herein the “HNF3beta”, or “HNF3b”, used interchangeably areintended to mean hepatocyte nuclear factor 3, a transcription factorregulating gene expression in endodermal derived tissue, e.g. the liver,pancreatic islets, and adipocytes. HNF3beta may sometimes also bereferred to as HNF3b or Fox2A the latter name originating from thetranscription factor being a member of Forkhead box transcriptionfactors family.

As used herein the term “OCT-1” is intended to mean organic cationtransporter 1. OCT-1 is a major hepatic transporter that mediates theuptake of many organic cations from the blood into the liver where thecompounds may be metabolized or secreted into the bile.

As used herein the term “MDR” is intended to mean multi-drug resistancetransporter. MDR 1 and 3 are members of the ATP-binding cassette (ABC)family of transporters and both are drug efflux transporters. MDR 1 isimportant in regulating the traffic of drugs, peptides and xenobioticsinto the body and in protecting the body against xenobiotic insults anddrug toxicity, while MDR 3 is essential for phospholipid secretion intobile.

As used herein the term “Activin” is intended to mean a TGF-beta familymember that exhibits a wide range of biological activities includingregulation of cellular proliferation and differentiation such as“Activin A” or “Activin B”. Activin belongs to the common TGF-betasuperfamiliy of ligands.

As used herein the term “xeno-free” is intended to mean completecircumvention of direct or in-direct exposure to non-human animalcomponents.

As used herein, the term “hepatocellular toxicity” indicates cellularresponses such as necrotic toxicity, apoptosis, mitochondrial toxicity,phospholipidosis, steatosis and bile acid transport.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Overview of the derivation protocol of hepatocyte-like cellsfrom hPS, as further outlined in example 2 and 3. The striped bardescribes the times for addition of a GSK3 inhibitor.

FIG. 2. Detailed variants of the protocols for differentiating hPStowards hepatocyte-like cells, protocol i to iv, as further described inexamples 4-11.

2 i. shows the control culture conditions without GSK-3 inhibitor added.

2 ii. shows one aspect of the invention, in which the GSK-3 inhibitor isadded to the growth medium after initial differentiation, hence when thecells are showing characteristics similar to type of the endodermal ormore specifically definitive endoderm lineage. In this aspect, the GSK-3inhibitor is removed when the cells are showing characteristic similarto hepatic progenitor cells.

2 iii. shows one aspect of the invention, in which the GSK-3 inhibitoris added to the growth medium after differentiation into hepatocyteprogenitors. Thus in this aspect of the invention, the GSK-3 inhibitoris added when the cells are showing characteristic similar to hepaticprogenitor cells.

2 iv. shows one aspect of the invention, in which the GSK-3 inhibitor isadded to the growth medium after initial differentiation when the cellsare showing characteristics similar to type of the endodermal or morespecifically definitive endoderm lineage. In one further aspectaccording to iv. the type and concentration of the GSK-3 inhibitor maybe changed during the culturing period.

FIG. 3. Further variants of protocols for differentiating hPS towardshepatocyte-like cells showing media and stages in the protocol ofinducing hPS to hepatocyte-like cells. A) shows the control cultureconditions without a GSK inhibitor. B) Shows the addition of a GSK3inhibitor at day 14, or when the cells resemble cell of the hepaticprogenitor type. C) Shows the use of a split media (SM) before theaddition of the GSK3 inhibitor. The protocols are further described inexamples 12-14.

FIGS. 4A-C. Feeder free cultured hPS cells with and without the GSK3inhibitor, as well as an alternative method where a split media asdescribed in FIG. 3C were compared for their hepatic profile andhomogeneity.

A) Results from Activity assay of CYP1A and CYP3A measured by conversionof paracetamol and OH_midazolam respectively. The use of a split mediashows that high levels of CYP3AX can be maintained when a GSK3 inhibitorand a split media is used.

B) Shows a possible increase in proliferation by addition of a GSK-3inhibitor, measured by an increase of the progenitor marker CD44 when aGSK3 inhibitor was added. The split media (SM) gave the same levels aswithout split media. Two controls were used undifferentiated cellscultured feeder free (UD feeder free) and a perpetual cell line from ahepatocellular carcinoma (HepG2). The levels of AFP, KRT18 (Keratin 18)and KRT19 (Keratin 19) indicate that the cells are maintained in a stagewhere they still have the ability to proliferate. The cells were 23 dayswhen they were analyzed, n=3.

C) Immunolocalisation of Beta-catenin and Dapi staining of hES-HEP-BIO(A) and hES-HEP+BIO (B) Beta-catenin is localized at the cell membranein hES-HEP cultures not treated and treated with BIO. Beta catenin inthe cytoplasm and nuclei is commonly observed in BIO treated hES-HEPcultures. Day 21, 20×

FIGS. 5A and B. hiPS cells cultured with and without the GSK3 inhibitorwere compared for their hepatic profile and homogeneity. Where a GSK3inhibitor was added it was added at a later stage of thedifferentiation, according to FIG. 3. Derivation of hepatocytes fromhuman induced pluripotent stem cells (hiPS).

A) Results from Activity Assay of CYP1A, 3A and 2C. The addition of aGSK3 inhibitor gave increased activity of CYP1A and CYP3A. The levels ofCYP2C are high in relation to what is usually shown for hepatocyte-likecells derived from hPS cells. The cells were 30 days, n=8. hiPS vshiPS+BIO.

B) Results of hepatic markers from Q-PCR of the same cells that wereanalysed by Activity assay (Q-PCR hiPS+/−BIO) n=4. The results show aclear increase of CYP1A1, CYP1A2, CYP3A4 and CYP7A1. The levels ofCYP2C9 where maintained high for the hiPS cells when a GSK inhibitor wasadded. The decreased AFP levels indicate maturation. Albumin wasmaintained at a level high compared to known protocols for thederivation of hepatocyte-like cells from hPS cells. AAT was maintainedon a high level. Important transporters such as MRP2, Oct-1, GSTA1, BCEPand OATP2 where maintained at levels significantly higher than HepG2showing a maturation of the hiPS cell derived hepatocyte-like cells.

FIGS. 6A and B. hES cells cultured with and without the GSK3 inhibitorwere compared for their hepatic profile and homogeneity, as described inexamples 12 and 13. Where a GSK3 inhibitor was added it was added at alater stage of the differentiation, according to FIG. 3B.

A) Results from Activity Assay of CYP1A, 3A and 2C. The addition of aGSK3 inhibitor gave increased activity of CYP1A, 3A and 2C. The cellswere 30 days, n=8. hPS vs hPS+BIO.B)

B) Results of hepatic markers from Q-PCR of the same cells that wereanalysed by Activity assay (Q-PCR hPS+/−BIO) n=4. The results show aclear increase of CYP1A1, CYP1A2, CYP3A4, CYP2C9 and CYP7A1. An increasein AAT was shown when BIO was added. Important transporters such asMRP2, Oct-1, GSTA1, BCEP and OATP2 where maintained at levels showing amaturation of the hPS cell derived hepatocyte-like cells.

FIGS. 7A-C. Induction of Cyp1A by GSK3β inhibitor in hESC derivedhepatocytes.

A) Shows Cytochrome P450 activity of CYP1A in hESC-HEP differentiatedwith and without GSK3β inhibitor (Example 4 (MMI-BIO) vs. Example 8(MMI+BIO) vs. Example 9 (MMII+BIO)). Analysis are performed at day 16-18(n=7-8), 20-21 (n=5) and 25 (n=2) respectively, mean±SD. The tables listthe increase of CYP activity as a fold change value.

B) Show gene expression levels of CYP1A1 and CYP1A2 respectively inhepatocyte-like cells differentiated in the presence of GSK3 inhibitor(Example 9) compared to HepG2. Analysis are performed day 16-18 (n=5),19-21 (n=4) for CYP1A2 and day 16-19 (n=14), 21-23 (n=6) for CYP1A1,mean±SD. (Cells obtained as described in example 9 vs. HepG2 cells vs.hPS cells)

C) show immunocytochemistry of CYP1A2 (red) at day 19 in hepatocyte-likecells differentiated in the presence of GSK3β inhibitor (Example 9) . .. .

FIGS. 8A-E. Differentiation of hESC derived hepatocytes in the presenceof GSK3 inhibitor from day 3 purifies hepatocytes from other cell types.

A-C show hESC-HEP at day 17, differentiated by protocol i or iiaccording to FIG. 2. D-E show hESC-HEP at day 15, differentiated byprotocol iii or vi according to FIG. 2,

A) 0 μM BIO, protocol i.

B) 1 μM BIO day 3-9, protocol ii.

C) 5 μM BIO day 3-9, protocol ii.

D) 0 μM BIO day 3-9, 1.5 μM BIO day 10-15 protocol iii,

E) 3.5 μM BIO day 3-9, 1.5 μM BIO day 10-15 protocol vi.

A and B show hESC-HEP cultures which are over grown by another cell typewhere as in C the majority of cells in the culture are hepatocyte-likecells. E represent a purer hESC-HEP culture than D. White arrow:hepatocyte-like cells, black arrow: other cell type than hepatocyte-likecells. Scale bar: 100 μm.

FIG. 9. Functional CYP1A activity in hES-HEP cultures differentiated inmedia supplemented with NaB (an Histone deacetylase inhibitor (HDAC)inhibitor) and BIO (a GSK3 inhibitor), compared to cultures without NaBin the maturation media, suggesting HDAC inhibitors, eg NaB, topotentiate Wnt-signalling mediated transcription. Analysis is performedday 24-25, n=1.

FIGS. 10A-D. Gene expression levels of hepatic markers are induced byBIO supplemented at day 3. Qrt-PCR data is presented as fold change geneexpression levels of HepG2 except for BSEP, which graph show fold changeof calibrator. N=2-3, mean±SEM. FIGS. 10A-D corresponds to example 22.

A) Phase I, drug metabolising enzymes: CYP3A4, CYP3A5, and CYP2C9 showincreased expression levels at the addition of BIO. CYP3A7 shows nosignificant increase

B) Phase II, drug metabolising enzymes: GSTA1 and UGT2B7 shows andincreased expression when BIO was added.

C) Phase III, transporters MRP2 and BSEP shows both increased expressionwhen BIO was added.

D) General liver markers: A1AT showed a strong increase at day 28. ALBdecreased at day 26 indicating maturation of the hepaticyte-like cells.TAT showed higher expression levels than for the cells cultured with BIOthan the ones with out BIO for all days.

FIG. 11. Relative expression levels of UGT hepatic metabolic markers inhESC-HEP generated with or without Wnt-signalling modulation by GSK-3inhibitor BIO during endoderm to hepatocyte-like cell phase. FC=foldchange relative expression, control set 30 to 1 for each example. Graphsshow expression levels in cells treated with one of threedifferentiation/maturation protocols (see example 21) and +/−GSK-3inhibitor treatment for activity of UGTs on specific UGT substrates:(UGT1A1,[8-estradiol]); (UGT2B, [8-estradiol]; (UGT1A6, [1-Napthol]),(UGT1A9, [Propofol]), (UGT2B7, [Naloxone]). A non-specific control(Methylumbelliferone) was also included. n=number of experimentsaveraged to obtain results.

FIGS. 12A and B. Relative expression levels of hepatic markers inhESC-HEP generated with modulation of Wnt-signalling by GSK-3 inhibitorsother than BIO during later maturation period (day 10 onwards).Treatments shown include: hESC-HEP (negative control; cells neverexposed to GSK-inhibitor during differentiation), BIO, SB216763,Kenpaullone, Indirubin-3-O; also included is a second negative controlundifferentiated hESC cells (hESC). FIG. 12A shows expression of Phase Ienzymes, FIG. 12B shows expression of Phase II enzymes and hepaticmarkers. GSK-3 inhibitors were present only during later differentiation(from day 10 onwards); also included is a further negative control graphshowing the expression levels of a housekeeping gene (Creb) across thevarious samples.

FIGS. 13A and B. Relative expression levels of hepatic markers inhESC-HEP generated with modulation of Wnt-signalling by GSK-3 inhibitorsother than BIO during both mid (days 3-9) and late stage (days 10-23)differentiation. Treatments shown include: hESC-HEP (negative control;cells never exposed to GSK-inhibitors), BIO, SB216763, Kenpaullone,Indirubin-3-O; also included is a second negative controlundifferentiated hESC cells (hESC); also included is a further negativecontrol graph showing the expression levels of a housekeeping gene(Creb) across the various samples. FIG. 13A shows expression of Phase Ienzymes, FIG. 13B shows expression of Phase II enzymes and hepaticmarkers.

FIGS. 14A-C. Relative expression levels of Phase I enzymes (A), Phase IIenzymes (B) and General hepatic markers (C) in hiPS-HEP cells generatedwith modulation of Wnt-signalling by GSK-3 inhibitors other than BIOduring later (day 10+) differentiation. Treatments shown include:iPS-HEP (negative control; cells never exposed to GSK-inhibitors), BIO,SB216763, Kenpaullone, Indirubin-3-O; also included is a second negativecontrol undifferentiated hiPS cells (iPS).

FIGS. 15A-C. Relative expression levels of Phase I enzymes (A), Phase IIenzymes (B) and General hepatic markers (C) in hiPS-HEP cells generatedwith modulation of Wnt-signalling by GSK-3 inhibitors other than BIOduring both mid (days 3-9) and late stage (days 10-23) differentiation.Treatments shown include: iPS-HEP (negative control; cells never exposedto GSK-inhibitors), BIO, SB216763, Kenpaullone, Indirubin-3-O; alsoincluded is a second negative control undifferentiated hiPS cells (iPS).

EXAMPLES

In present invention, several modulators of the Wnt pathway have beentested, including GSK inhibitor BIO (GSK inhibitor IX), Kenpaullone,SB216763 and Indirubin-3′-oxime. As well known within the field anddiscussed in the scientific literature as example (Nejak-Bowen et al2008), the other GSK-3 inhibitors and other molecules effecting thesignalling cascade are suggested to have a similar effect for modulationof Wnt signalling pathway. Examples of general culturing and passagingtechniques are disclosed in pending applications PCT/EP2004/005033,PCT/EP02/14895, PCT/EP2005/040582, PCT/EP2006/009697, PCT/EP2007/004940and PCT/EP208/059491.

As laid out in the examples, the starting material may comprise anypluripotent stem cell derived through an initial differentiation towardsa definitive or extraembryonic lineage. The starting material may alsobe any cell of hepatic progenitor lineage.

Example 1 Starting Material for Hepatocytes Derived from HumanPluripotent Stem Cells Maintained on Feeder Cells

All hPS cells (as defined above) can be used as staring material forthis invention. For the examples below in particular hepatocyte-likecells were derived in vitro from undifferentiated human embryonic stemcells (hESC) cultured on mEF cells (Heins et al 2004, Stem Cells). Thecell lines used for this experiment could be, but is not limited to thehES cell line SA002, SA121 and SA181 (Cellartis AB, Göteborg, Sweden)and they can be propagated as described Heins et al. 2004. These celllines are listed in the NIH stem cell registry, the UK Stem Cell bankand the European hESC registry and are available on request. Along withhPS obtained from hESC, hPS cells invention hPS obtained from hiPS(induced pluripotent stem cells) have been used for the derivation ofhepatocytes for the examples of this invention.

Example 2

Derivation of hepatocytes from human pluripotent stem cells using a GSK3inhibitor. Hepatocytes were derived from both hES cells and human hiPScells according to the protocol in FIG. 1, this protocol gives anoverview of the derivation of human hepatocyte-like cells from humanpluripotent stem cells.

Before adding the first medium, ID day 0-2, the cultures were washedthoroughly with PBS, twice. The different mediums were prepared freshlyand added day 0 (ID day 0-1), 2 (ID day 2-4), 4 and 7-10 every second orthird day (VH1), 10-28 every second or third day (MMI or MM II). Cellsare passaged at day 4 and replated at a cell density of 50 000-350 000cells/cm² such as e.g. 100 000-300 000 cells/cm², preferably 200 000cells/cm².

The Initial Differentiation (ID) Step

Day 0-1

RPMI 1640 (+0.1% PEST, +1% Glutamax)

1×B27

100 ng/ml Activin A

1 mM NaB

Day 2-3

RPMI 1640 (+0.1% PEST+1% Glutamax)

1×B27

100 ng/ml Activin A

0.5 mM NaB

Hepatic Progenitor Step

Day 3

+/−3.5 μM GSK-3 inhibitor (e.g. BIO)

VH1

Day 4-9

VitroHES

1% DMSO

+/−3.5 μM GSK-3 inhibitor (e.g. BIO)

Maturation media (MM) I

Day 10-30

WME+SQ (GA1000)+1% Glutamax+0.1% PEST)

10 ng/ml OsM

0.1 μM DexM

2 ng/ml bFGF

10 ng/ml HGF

0.5% DMSO

10 mM Nicotinamide

ITS (10 μl/ml)

3 ng/ml Glucagon

+/−1.5 μM GSK-3 inhibitor (e.g. BIO)

Example 3

As outlined in Example 2 and in FIG. 1, but with maturation medium II(MM II) replacing maturation medium I (MM I).

Maturation Media (MM)

Day 10-30

WME+SQ (GA1000)+1% Glutamax+0.1% PEST)

10 ng/ml OsM

0.1 μM DexM

20 ng/ml HGF

0.5% DMSO

+/−1.5 μM GSK-3 inhibitor (e.g. BIO)

Example 4

As Example 2, but without addition of a GSK3 inhibitor. Example 4 is acontrol protocol in which hESC were differentiated into hepatocyte-likecells in the absence of GSK3 inhibitor.

As outlined in FIG. 2) (i-iv), variants of the overview protocol in FIG.1 were tested. The differentiation procedure follows three stages, firstduring the initial differentiation (ID) step, partly differentiatedcells resembling endodermal cells are formed (day 0-4), second thepartly differentiated cells are differentiated into hepatoblasts/hepaticprogenitors (day 4-10) and finally the hepatoblasts are matured intohepatocyte-like cells (day 10-30).

Example 4 was performed as schematically depicted in FIG. 2 i.

Example 5

As example 3, but without addition of a GSK3 inhibitor. Included as acontrol protocol of which hESCs were differentiated into hepatocyte-likecells in the absence of GSK3 inhibitor.

Example 5 was performed as schematically depicted in FIG. 2 i.

Example 6

Schematically depicted in FIG. 2 ii. As example 2 but with 3.5 μM GSK3inhibitor is added at day 3 to day 10 only.

Example 7

Schematically depicted in FIG. 2 ii. As example 3 but with 3.5 μM GSK3inhibitor is added at day 3 to day 10 only.

Example 8

Schematically depicted in FIG. 2 iii.

As example 2 but with 1.5 μM GSK3 inhibitor is added at day 10 to day 30only.

Example 9

Schematically depicted in FIG. 2 iii.

As example 3 but with 1.5 μM GSK3 inhibitor is added at day 10 to day 30only.

Example 10

Schematically depicted in FIG. 2 iv. As example 2 but with 3.5 μM GSK3inhibitor added at day 3 to day 10 and changed to 1.5 μM GSK3 inhibitorat day 10 and throughout the protocol.

Example 11

Schematically depicted in FIG. 2 iv. As example 3 but with 3.5 μM GSK3inhibitor added at day 3 to day 10 and changed to 1.5 μM GSK3 inhibitorat day 10 and throughout the protocol.

Example 12 Medium and Supplement Factors for Hepatocyte-Like CellsDerived from Feeder Free hES or hiPS

Wash the cells prior initiation of differentiation of theundifferentiated hPS hepatocyte-like cells. Before adding the firstmedium, Initial differentiation (ID) step day 0-2, the undifferentiatedcultures (UD) in T150 flasks were washed thoroughly with PBS or RPMI640,twice. The different mediums were prepared freshly and added daily atday 0 (ID day 0-1), 2, 3 and 4 (ID day 2-4), 4. Then the cells werepassaged at a concentration of approximately 200,000 cells/cm² tofreshly gelatine- or matrigel coated 24 well plates in VH1 medium. Thematuration medium was then changed every second day or third day for day7-10, and for day 10-28 every second or third day (BM2 or ModII).

Example 12 illustrates the derivation without a GSK3 inhibitor (BIO)used as a control for this invention and was carried out as outlined inFIG. 3 A).

The Initial Differentiation (ID) Step

Day 0-1

RPMI 1640 (+0.1% PEST, +1% Glutamax)

1×B27

100 ng/ml Activin A

1 mM NaB

ID

Day 2-7

RPMI 1640 (+0.1% PEST)

1×B27

100 ng/ml Activin A

0.5 mM NaB

Day 7 the cells are passaged with TrypLE Select. The cells are incubatedfor 3-7 minutes at 37° C. Diluted and washed with VH medium, spun at 300g, 5 min. Thereafter, the cells were seeded onto fresh coated dishes.

VitroHES 1 Step (VH1)

Day 7-14

VitroHES™ (VH)

1% DMSO

Maturation Media BM2 (or Alternatively MMII (as Described in Example 2))Day 14-28

WME+SQ (-GA1000, +1% Glutamax+0.1% PEST)

(10 ng/ml OsM)

0.1 μM DexM

10 ng/ml HGF

0.5% DMSO

Example 13

Performed as outlined in FIG. 3 B). As example 7 but with 1.4 μM GSK3inhibitor is added at day 14.

Example 14

Performed as outlined in FIG. 3 C) describes the derivation with thecells cultured in a split medium between day 7-9 and a GSK3 inhibitorintroduced at day 14.

The Initial Differentiation (ID) Step

Day 0-1

RPMI 1640 (+0.1% PEST, +1% Glutamax)

1×B27

100 ng/ml Activin A

1 mM NaB

ID Day 2-7

RPMI 1640 (+0.1% PEST)

1×B27

100 ng/ml Activin A

0.5 mM NaB

Day 7 the cells are passaged with TrypLE Select. Incubated for 4 minutesat 37° C. Diluted and washed with VH4 medium, spun at 300 g, 5 min.Thereafter, the cells were seeded onto fresh coated dishes.

Split Media (SM) Day 7-9

RPMI A (+0.1% PEST+1% Glutamax (10 μl/ml)

100 ng/ml aFGF

5 ng/ml bFGF

50 ng/ml BMP2

200 ng/ml BMP4

0.2% FBS

VitroHES 1 Step (VH1) Day 9-14

VitroHes

1% DMSO

Maturation Media BM2 (or Alternatively MMII (as Described in Example 2))Day 14-28

WME+SQ (-GA1000, +1% Glutamax+0.1% PEST)

(10 ng/ml OsM)

0.1 μM DexM

2 ng/ml bFGF

10 ng/ml HGF

0.5% DMSO

10 mM nicotineamide

10 μg/ml ITS

3 ng/ml Glucgon

1.4 μM BIO

Example 15 Induction of Cytochrome P450 1A in mEF-Cultured hESC DerivedHepatocytes by GSK3-Inhibitor

MEF-cultured hESCs (hES cells cultured on feeder cells) weredifferentiated into hepatocyte-like cells according to examples 4, 8 and9, thus comparing the absence of BIO, a GSK3-inhibitor (Example 4), tothe influence of BIO during the maturation step (day 10-26) inmaturation medium I and II (MM I and MM II)(Example 8 and 9respectively). Before adding the first medium, ID day 0-2, the cultureswere washed thoroughly with PBS, twice. The different mediums wereprepared freshly and added day 0 (DE day 0-1), 2 (DE day 2-4), 4 and7-10 every other to third day (VH1), 10-26 every second or third day(MMI or MM II). Detailed information about the composition of thedifferent mediums, see examples 4, 8 and 9. At day four, the cells werepassaged to new dishes in order to obtain a confluent layer of cellsthat subsequently was differentiated into hESC-HEP. Briefly, the cellswere detached from the culture unit by incubating the cells in an enzymesolution, Tryple Select, for 5 to 10 min. VitroHes-medium was added tothe cultures to stop the effect of the enzyme. The detached cells weretransferred to tubes and centrifuged for 5 min at 300 g. The supernatantwas discarded and VH1-medium added to the cell pellet, which wassubsequently dissociated into single cell suspension. Cells were countedin a Bürker chamber and seeded out in 0.1% gelatine coated culture units(e.g. in 24-well plates) at a cell density of 150 to 250K cells per cm².

At day 16, 18, 20, 21 and 25 hESC-HEP cultures were analyzed forcytochrome P450 1A activity by incubating the substrate Phenacetine to afinal concentration of 26 μM in Phenol Red-free Williams Medium E,supplemented with 0.1% Penicilline-Streptomycin 2 mM L-Glutamine and 25mM Hepes. A volume of 220 μl diluted substrates were added per well of a24-well plate. hESC-HEP cultures with substrates were incubated overnight. After 16 h, medium was collected and subsequently, centrifuged at10 000 g, 4° C. for 20 min. Samples were analysed by Liquidchromatography-mass spectrometry (LC-MS) LCMS for presence of themetabolite Paracetamol, biotransformed by the cytochrome p450 enzymesCyp1A2, 1A1.

Results

hESC-HEP, matured in maturation medium I supplemented with 1.5 μM BIO(MMI+BIO) according to Example 8, were able to metabolize phenacetineinto paracetamol to a greater extent than the control cultures accordingto example 4, see FIG. 7A. On top of that, hES-HEP cultured inmaturation medium 11 supplemented with 1.5 μM BIO (MMII+BIO)(example 9)performed even better than MMI+BIO cultures regarding Cyp 1A activity.This trend was demonstrated for all time points analysed, day 16-18,20-21 and day 25. The Cyp1A activity increased during time in the twogroups treated with BIO, suggesting maturation of hepatocyte-like cellsover time in the presence of a GSK3 inhibitor. The Cyp 1A activity wassupported by the finding of CYP1A2 and 1A1 gene expression levelssimilar to or greater than HepG2, see FIG. 7B. In addition, the proteinCYP1A2 was detected in hESC-HEP cultures matured in the presence of BIO,see FIG. 7C. To summarise GSK3 inhibitors stimulate CYP1A-family membersto be functionally expressed at both mRNA and protein levels inhESC-HEP. As CYP1A1 is expressed in the neonatal liver and CYP1A2 in thenewborn and adult liver, the results point at GSK3 inhibitors to take animportant part in differentiation and maturation of hESC intohepatocyte-like cells.

Example 16 hPS Cells from Feeder Free Cultures

hESCs cultured under feeder free conditions were incubated in mediasupplemented with Activin A. The cells were then induced intohepatocytes by differentiating them to hepatic progenitor cells and thento more mature hepatocyte like cells. Cells cultured with and withoutthe GSK3 inhibitor were compared for their hepatic profile andhomogeneity. For culture details see FIG. 3 and examples 12, 13 and 14.

From this study we could conclude that a GSK3 inhibitor (BIO) wassignificantly important for the differentiation, maturation, andhomogeneity of the cultures. (See results in FIGS. 4A & B), since bothmetabolic activity (FIG. 4A) and hepatic gene marker expression (FIG.4B) was higher in cells exposed to GSK-3 inhibitors and thus comparablewith hESC-HEPs derived from hPS initially maintained on feeder cells.Confirmation that BIO treatment is affecting the Wnt pathway shown inFIG. 4C where beta-catenin is seen to translocate from cell membrane tonucleus upon with BIO, consistent with its signalling role in the Wntpathway.

Example 17 Derivation of Hepatocyte Like Cells from hiPS Cells

The culturing and derivation was performed as described in example 12-14and outlined in FIG. 3, but with hiPS cells replacing the feeder freehPS cells.

The undifferentiated hiPS cells were cultured in Activin A supplementedmedia to stimulate initial differentiation into partly differentiatedcells. The hiPS derived partly differentiated cells were then passaged(to plates coated with 0.1% Gelatin or Matrigel 0.016 mM) and induced tohepatic progenitor cells and then to hepatocyte like cells in media withand without BIO supplementation, see FIG. 3 A)-B).

The conclusion from this study was that the hiPS cells cultured in mediasupplemented with BIO responded significantly and became more maturecompared to the cells that were grown in the absence of the GSK-3inhibitor. This was concluded by analysing the expression profile of thecells by Q-PCR, immunocytochemistry and Activity Assay FIGS. 5A and B.These results verify and are unanimous with what we have observed forboth hES cultured on mEF and hES cultured in a feeder free way (FIGS. 4Aand B)

The hiPS cells were cultured on mEFs until they were confluent. Thecells were washed twice in PBS+/+ and treated with Activin A containingmedium (See FIG. 3 a-b). The cells adapted an endoderm like morphologyaround day 3. When the majority of the cells were partly differentiated,the cells were exposed to media supplemented with factors inducing thecells into hepatocyte-like cells (See FIG. 3A)-B)) plus or minus BIO.Different matrix: Gelatin and Matrigel did not significantly affect theoutcome. BIO affected the cells significantly in that they upregulatedseveral markers for mature hepatocytes including CYPs (CYP1A2) (seeFIGS. 5A and B). hESC-HEPs treated with BIO also showed greatermetabolic activity (FIG. 5A).

Results

Conclusions of BIO in the Maturation Phase of the Hepatocyte-Like CellProtocol:

-   -   Increased CYP1A activity compared to controls without BIO.    -   Increased mRNA of CYP1A1, CYP1A2, CYP3A4, CYP2C9, CYP7A1, MRP2,        CD44, AFP, CK18, CK19 activity compared to controls without BIO.

Conclusions of the use of a split medium (SM) and BIO in maturationphase of the hepatocyte-like cell protocol as of FIG. 3c ).Illustrations of the results are shown in FIGS. 5A and B

-   -   Increased CYP1A and CYP3A activity compared to controls without        BIO.    -   Increased mRNA of CD44, AFP, CK18, CK19, CYP1A1, CYP1A2, CYP3A4,        CYP2C9, CYP7A1, Albumin, OATP2, A1AT, MRP2 activity compared to        controls without BIO.

Example 18 Selective Differentiation Using a GSK3 Inhibitor (BIO)Results in a Homogenous Population of Hepatocytes Derived from hES Cells

MEF cultured hESC differentiated into hepatocyte-like cells according toexample 6 and 11 as outlined in FIG. 2 ii and iv (presence of BIO fromday 3) was compared to hepatocyte-like cells differentiated by example 4and 8D as outlined in FIG. 2 i and iii (absence of BIO and with BIOadded from day 10-30, respectively) demonstrated purification ofhepatocyte-like cells in the cultures treated with BIO from day 3(example 6 and 11). Morphological observations are illustrated in FIGS.8A-E. Cultures with no or low concentrations (1 μM) of BIO resulted inhighly heterogeneous cultures where hardly any hepatocyte-like cellswere observed as shown in FIGS. 8A and B. Addition of 5 μM of BIO day 3resulted in a dramatic cell death to begin with. However, the survivingcells, DE-cells and/or anterior endodermal cells, day 4 were passagedinto new wells and a highly purified and homogenous culture of hESC-HEPwas appearing as the differentiation process proceeded, see FIG. 8C. Anintermediate concentration of BIO (3.5 μM) was tested in cultures fromday 3 (Example 11) resulting in more purified hESC-HEP compared tountreated cultures (Example 9), FIGS. 8D and E respectively. Inaddition, a less dramatic cell death was observed compare to culturesgrown in 5 μM BIO from day 3. Thus it appears advantageous to add GSK-3inhibitors at a later stage of differentiation to avoid massive celldeath seen when it is added at day 3. Moreover the resulting purity ofthe cell populations (FIGS. 8A-E, purity table) also suggest that, atleast for BIO, addition at a later stage (day 10+) gives greater finalpurity. Data suggest a role for GSK3 inhibitors in selection of DE-cellsand/or anterior endodermal cells, a prerequisite for furtherdifferentiation into hepatoblasts and subsequently hepatocyte-likecells. In addition, GSK3 inhibitors at this stage maycontribute/stimulate to hepatic induction of the competent endoderm.

Example 19 HDAC Inhibitors Potentiates Wnt-Signalling to Induce andStimulate Hepatocyte Differentiation

FIG. 9 shows data of functional CYP1A activity in hESC-HEP cultures(hepatocyte-like cells) differentiated in a maturation media, such asMMI, supplemented with an HDAC inhibitor, such as Sodium Butyrate (NaB,1 mM) together with a GSK3 inhibitor, such as BIO (1.4 μM) at day 21-25,following the protocol as described in example 14. Cell line SA002 frommEF cultures was used as starting material.

Those hESC-HEP cultures were compared to parallel hESC-HEP cultureswithout NaB in the maturation media resulting in increased CYP1Aactivity in NaB containing cultures. Data suggests a role for HDACinhibitors, e.g. NaB, to potentiate Wnt-signalling mediatedtranscription and effect on hepatocyte differentiation.

Example 20 Exposure of GSK3 Inhibitor at Early Hepatic DifferentiationImproves Hepatic Gene Expression Profile of hESC-HEP

hESC-HEP were derived from cell-line SA002 cultures on mEF-layeraccording to Example 9, FIG. 2, protocol iii (absence of BIO between day3-10) or Example 11, FIG. 2, protocol iv (presence of 3.5 μM BIO betweenday 3-10). Total RNA was collected and isolated from the two hES-HEPcultures at day 21, 24, 26 and 28 by using RNA isolation kit fromQiagene. Quantitative reverse transcriptase PCR, QrtPCR, by using Taqmanprobes, was performed for the following hepatic marker genes: phase Idrug metabolizing enzymes; CYP (cytochrome P450) 3A4, 3A5, 3A7, 2C9,phase II drug metabolizing enzymes GSTA1 (glutation-S-transferas A1),UGT2B7 (UDP glucuronosyltransferase 2B7), phase III, transporters; MRP2(multi-drug residence protein 2), BSEP (bile salt export pump), andgeneral hepatic markers; A1AT (alpha-1 antitrypsin), ALB (albumin) andTAT (tyrosine-amino transferas). All data was normalised to thehouse-keeping gene CREB. RNA from HepG2 cultures was included and datais presented as fold change of HepG2. BSEP is an exception as HepG2cells do not express the gene. Thus, BSEP expression is insteadpresented as fold change of a so called calibrator which contains RNAfrom different sources.

Data is presented in FIGS. 10A to D and shows that all genes except forCYP3A7 are expressed at higher levels in hESC-HEP exposed to BIO fromday 3 than in cultures where BIO was excluded from day 3-10. For CYP3A7the opposite was observed. As CYP3A7 is a drug metabolizing enzyme whichis mainly expressed prenatal and CYP3A4 in the newborn and adult liver,(Cyp 3A5 both pre and post natal and adult) the expression pattern ofthe CYP3A family members suggests a role for BIO early in thedifferentiation protocol to improve hepatic differentiation andcontribute to a more maturation hESC-HEP culture by stimulating Wntsignalling. The improved expression levels of the hepatic markers in BIOexposed cultures are supporting the finding that GSK-3 inhibitors areimportant in early hepatic differentiation of hESC.

Example 21 Improvement of UGT Metabolic Activity in hESC-HEPs Exposed toGSK-3 Inhibition During Early Hepatic Differentiation

Metabolic activity of hESC-HEP derived from hESCs cultured infeeder-free conditions (see Examples 12-14) was measured to determinethe effect of GSK-3 inhibition during development and maturation byexamining the activity of several UDP-glucuronyltransferases (UGTs)(enzymes that participate in the metabolism of many drugs). Activity ofthese UGTs was tested via several substrates, namely: β-estradiol(UGT1A1, 3-glucuronide), 1-naphthol (UGT1A6), propofol (UGT1A9), andnaloxone (UGT2B7). A non-specific control (Methylumbelliferone) was alsoincluded (see FIGS. 12A-B). hESC-HEPs were differentiated essentiallyaccording to Example 12, up to day 7. At this point, cells were treatedto one of three protocols, either Protocol 1 (day 7-14 VH1 medium, day14-25 Maturation medium BM2, BIO present only at days 14-25), Protocol 2(day 7-25 Medium BM2, BIO present only at days 14-25) or Protocol 3 (day7-14 Medium MMI, day 14-25 Maturation Medium MMII, BIO present days14-25) with either +/−GSK-3 inhibitor (in this case BIO) at aconcentration of 1.4 μM. A clear trend can be seen here, with most ofthe UGTs having greater activity in cells treated with GSK-3 inhibitorduring development, and this trend is seen across all threedifferentiation protocols. For some UGTs (such as UGT1A1) the increaseis much greater than for others, but the overall trend is that exposureof cells to GSK-3 inhibition leads to a more mature, metabolicallyactive phenotype for all of the preferred differentiation methods.

Example 22 Induction of Early and Late Stage Hepatic Markers inhESC-HEPs by Modulation of Wnt-Signalling Using Non-810 GSK-3 Inhibitorsat Later Stage Differentiation

hESC-HEPs were derived essentially as described in Example 9, FIG. 2iii,but with alternative (non_BIO) GSK-3 inhibitors used to show that otherGSK-3 inhibitors can be used interchangeably with BIO and can modulatethe Wnt-signalling pathway during endoderm to hepatocyte differentiationto induce expression of hepatic marker genes. Furthermore,differentiating cells were exposed to GSK-3 inhibitors only during later(from day 10) stages of differentiation. Three alternative GSK-3inhibitors were tested alongside BIO to determine effectiveness ininducing hepatic marker expression (SB216763, Kenpaullone andIndirubin-3-O) and results were compiled both for Phase I and Phase IIenzymes and hepatic markers, with two negative controls (hESC-HEP notexposed to GSK-3 inhibition during differentiation, and undifferentiatedhESC cells). Results are shown in FIG. 12A (Phase I enzyme markers) andFIG. 12B (Phase II enzymes and hepatic markers), and it can be seen thatfor Phase I enzymes, all GSK-3 substitutes in general seem to inducegene expression above the levels of the negative controls. Indeed, formost of the Phase I enzymes and hepatic markers, Kenpaullone seems toinduce higher levels of expression than BIO. Results for Phase IIenzymes and hepatic markers are somewhat more variable when differentmarkers and different GSK-3 inhibitors are compared, but again ingeneral it appears that the three alternative compounds tested can beused as feasible substitutes for BIO in modulating the Wnt-signallingpathway and producing mature hepatocyte-like cells. Expression of thehousekeeping gene Creb shows little variation across the various samplesand protocols. Equivalent results where hiPS cells were used instead ofhESC-HEPS are shown in FIGS. 14A-C, and again support the finding thatBIO can be substituted by other GSK-3 inhibitors.

Example 23 Induction of Early and Late Stage Hepatic Markers inhESC-HEPs by Modulation of Wnt-Signalling Using Non-810 GSK-3 Inhibitorsat Both Mid and Later Stage Differentiation

hESC-HEPs were derived essentially as described in Example 11 and FIG.2iv, but with alternative (non_BIO) GSK-3 inhibitors used to show thatother GSK-3 inhibitors can be used interchangeably with BIO and canmodulate the Wnt-signalling pathway during endoderm to hepatocytedifferentiation to induce expression of hepatic marker genes.Differentiating cells were exposed to GSK-3 inhibitors during both mid(days 3-9) and late (from day 10-23) stages of differentiation. Threealternative GSK-3 inhibitors were tested alongside BIO to determineeffectiveness in inducing hepatic marker expression (SB216763,Kenpaullone and Indirubin-3-O) and results were compiled both for PhaseI enzymes and Phase II enzymes and hepatic markers, with two negativecontrols (hESC-HEP not exposed to GSK-3 inhibition duringdifferentiation, and undifferentiated hESC cells). During days 3-9 ofdifferentiation, concentrations of GSK-3 inhibitors used were asfollows: BIO 3.5 μM, SB216763 34 nM, Kenpaullone 0.23 μM andIndirubin-3-O 22 nM. For later stage differentiation (days 10-23)concentrations were changed to: BIO 1.5 μM, SB216763 2.5 μM, Kenpaullone2.5 μM and Indirubin-3-O 2.5 μM Results are shown in FIG. 13A (Phase Ienzymes) and FIG. 13B (Phase II enzymes), and it can be seen that forPhase I enzymes, GSK-3 substitutes can in general seem to induce hepaticmarker gene expression above the levels of the negative controls andoften above the level of that seen in BIO treatment. Indeed, for most ofthe Phase I enzymes, Kenpaullone seems to induce higher levels ofexpression than BIO. Results for Phase II enzymes and hepatic markersare somewhat more variable when different markers and different GSK-3inhibitors are compared, but again in general it appears that the threealternative compounds tested can be used as feasible substitutes for BIOin modulating the Wnt-signalling pathway and producing maturehepatocyte-like cells. It is also apparent that modulation ofWnt-signalling at mid and late stages by GSK-3 inhibition is comparableto modulation during only late stage. Depending upon the result desired(expression of certain markers over others) then one method may bepreferable over the other. Expression levels of the housekeeping geneCreb shows little variation across the various samples and protocolshere. Equivalent results where hiPS cells were used instead of hESC-HEPSare shown in FIGS. 15A-C, and again support the finding that BIO can besubstituted by other GSK-3 inhibitors.

The invention claimed is:
 1. A composition comprising: an in vitroderived hepatocyte-like cell in a media comprising one or more GSK3inhibitors, wherein said in vitro derived hepatocyte-like cell haselevated expression of the genes CYP1A1, CYP1A2, CYP3A4, CYP7A1, andMRP2 compared to a comparable hepatocyte-like cell obtained underconditions without use of any GSK3 inhibitor.
 2. The composition ofclaim 1, wherein said in vitro derived hepatocyte-like cell hasenzymatic activities exceeding a fold change of at least 10 incytochrome P450 activity when compared to saki comparablehepatocyte-like cell obtained under conditions without use of any GSK3inhibitor.
 3. The composition of claim 1, wherein said in vitro derivedhepatocyte-like cell has enzymatic activities exceeding a fold change ofat least 13 in cytochrome P450 activity when compared to saki comparablehepatocyte-like cell obtained under conditions without use of any GSK3inhibitor.
 4. The composition of claim 1, wherein said one or more GSK3inhibitors is selected from the group consisting of: BIO, kenpaullone,SB2167763, Indirubin-3′-monoxime, and any combination thereof.
 5. Thecomposition of claim 1, wherein said one or more GSK3 inhibitors is BIO.6. The composition of claim 1, wherein said one or more GSK3 inhibitorsis kenpaullone.
 7. The composition of claim 1, wherein said one or moreGSK3 inhibitors is SB2167763.
 8. The composition of claim 1, whereinsaid one or more GSK3 inhibitors is Indirubin-3′-monoxime.
 9. Thecomposition of claim 1, wherein said in vitro derived hepatocyte-likecell further has elevated expression of the gene CYP2C9 compared to thecomparable hepatocyte-like cell obtained under conditions without use ofany GSK3 inhibitor.
 10. The composition of claim 1, wherein said invitro derived hepatocyte-like cell further has elevated expression ofthe genes OATP2 and AAT compared to the comparable hepatocyte-like cellobtained under conditions without use of any GSK3 inhibitor.